ES2614880T3 - HER2 DNA vaccine as an auxiliary treatment for cancers in pets - Google Patents

Abstract

A xenogenic Her2 / neu antigen that is xenogenic of a Her2 / neu antigen expressed by mammary cells of a dog, for use in the treatment of canine mammary carcinoma / tumor in a dog suffering from canine mammary carcinoma / tumor, where the Her2 / antigen neu xenogeneic is in an immunologically effective amount and the Her2 / neu xenogenic antigen is administered after surgical removal of tumors in subjects suffering from such cancers, and where the Her2 / neu antigen associated with xenogenic mammary gland tumor is a vector comprising a DNA sequence encoding the Her2 / neu antigen associated with xenogenic therapeutic mammary gland tumor under the control of a promoter that promotes the expression of the Her2 / neu antigen associated with a mammary gland tumor in dogs.

Description

DESCRIPTION

HER2 DNA vaccine as an auxiliary treatment for cancers in companion animals 5 BACKGROUND OF THE INVENTION

[0001] This application relates to compositions for the treatment of differentiation of antigen-dependent cancers and to methods of using said compositions. The invention uses compositions containing xenogenic differentiation antigens that are associated with cancers to provide a

10 effective therapy.

[0002] Differentiation antigens are tissue-specific antigens that are shared by autologous and some allogeneic tumors of similar derivation and in counterparts of normal tissue at the same stage of differentiation. It has been shown that differentiation antigens are expressed by a variety of types

15 tumors, including melanoma, leukemia, lymphomas, colorectal carcinoma, breast carcinoma, prostate carcinoma, ovarian carcinoma, pancreatic carcinomas and lung cancers. For example, differentiation antigens expressed by melanoma cells include Melan-A / MART-1, Pmell7, tyrosinase and gp75. The differentiation antigen expressed by lymphomas and leukemia includes the differentiation markers of B lymphocytes CD 19 and CD20 / CD20 B. It is an example of a differentiation antigen expressed by colorectal carcinoma, breast carcinoma, pancreatic carcinoma, prostate carcinoma , ovarian carcinoma and lung carcinoma the mucin-1 mucin polypeptide. It is a differentiation antigen expressed, for example, by Her2 breast carcinoma (synonymous: Her2 / neu, ECBB2, ErbB2, c-erb-2), which is a gene that encodes a tyrosine kinase receptor that is a member of the family of epidermal growth factor receptors (De Maria et al., 2005). Her2 overexpression has been demonstrated in mammary gland tumors both in cats (Winston et al., 2005) and in dogs (Rungsipipat et al., 2008). Winston et al. (2005) used existing test procedures (HERcEpTEsT ™, Dako USA, Carpinteria, CA; NCL-CB11, Novocastra, Newcastle, UK) to successfully graduate Her2 expression levels in feline breast tumors as 0 = minimum / absent, 1 = weak, 2 = moderate or 3 = intense. The HERCEPTEST ™ and NCL-CB11 trials identified 27 and 23 cats respectively, of the 30 examined, for having a 2 or 3 degree of Her2 expression in breast tumor samples.

30

[0003] In addition to successfully graduating Her2 overexpression levels in mammary tumors of feline, Winston et al. (2005) used HERCEPTEST ™ to detect low levels of Her2 expression in normal feline epithelial tissues and cell types that include: hair follicle, mammary gland, gastric foveola, salivary gland duct, renal cortical and medullary tubules, colon crypt and of the small intestine, brain,

35 duct and pancreatic islets, splenic macrophages, adrenal cortex, hepatocytes and testicular Leydig cells. The expression of Her2 has been documented in a number of human epithelial cell types including gastrointestinal, respiratory, reproductive, urinary, cutaneous, mammary and placental (Press et al., 1990). These findings indicate that Her2 expression is common in a number of tissue types of humans and cats. The finding of Her2 overexpression in dog mammary tumors suggests that this species 40 share the expression characteristics identified in humans and cats. Existing assays and reagents can serve as tools to screen Her2 expression levels in companion animal cancers to justify treatment with Her2's cancer vaccine.

[0004] Unfortunately, in most cases, the individual's immune system is tolerant of said differentiation antigens and fails to create an effective immune response. Several have been considered

technologies to meet this challenge: (cytokines as genetic adjuvants (Chang et al., 2004), xenogenic vaccination (Pupa et al., 2005), electrotransfer (Quaglino et al., 2004), combination with chemotherapy (Bernhardt et al. , 2002) Although the results are encouraging, greater efficiency would be required for these approaches to be considered a key component of a therapeutic strategy.In addition, recent findings 50 indicate that both antibody and cell-mediated immunity are required for tumor eradication after of immunization, which perhaps explains the lack of success in the field (Orlandi et al., 2007) Therefore, for the treatment of cancers where the tumor expresses differentiation antigens, it would be desirable to have a response stimulation procedure Therapeutically effective immune system against differentiation antigen in vivo It is an object of the present invention to provide such a method.

55

References

[0005]

60 Orlandi et al. "Antibody and CD8 + T cell Responses against HER2 / neu Required for Tumor Eradication after DNA Immunization with a Flt-3 Ligand FusionVaccine." Clin Cancer Res 2007; 13 (20) October 15, 2007.

Amici A. et al. Venanzi FM, Concetti A. "Genetic immunization against neu / erbB2 transgenic breast cancer". Cancer

Immunol Immunother 1998; 47: 183-90.

Bergman PJ et al. "Long-Term Survival of Dogs with Advanced Malignant Melanoma after DNA Vaccination with Xenogeneic Human Tyrosinase: A Phase I Trial". CCR, Vol. 9, 1284-1290, April 2003.

Bargmann et al. "The neu oncogene encodes an epidermal growth factor receptor-related protein". Nature, 16-22 of January 5, 1986; 319 (6050): 226-30.

Norell H et al. "Vaccination with a plasmid DNA encoding HER2 / neu together with low doses of GM-CSF and IL-2 in patients with metastatic breast carcinoma: a pilot clinical trial". JTM, June 7, 2010, 8:53.

Jacob, JB et al. “Combining Human and Rat Sequences in Her2 DNA Vaccines Blunts Immune Tolerance and Drives Antitumor Immunity”; Cancer Res, January 1, 2010, 70; 119.

10 De Maria R et al. “Spontaneous Feline Mammary Carcinoma is a Model of Her2 Overexpressing Poor Prognosis Human Breast Cancer”; Cancer Res 2005: 65 (3); 907-912.

Philibert JC et al. “Influence of Host Factors on Survival in Dogs with Malignant Mammary Gland Tumors”; J Vet Intern Med 2003; 17: 102-106.

Press MF et al. "Expression of the Her2 / neu proto-oncogene in normal human adult and fetal tissues"; Oncogene, 5: 15 953-62.

Rungsipipat A et al .; "C-erbB-2 oncogene and P21WAF / CIPI tumor suppressor gene expression as prognostic factors in canine mammary adenocarcinomas"; Comp Clin Pathol 2008, 17: 35-41.

Winston J et al. "Immunohistochemical detection of Her = 2 / neu expression in spontaneous feline mammary tumors"; Veterinary and Comparative Oncology 3, 1, 8-15, 2005.

20 Chang sY et al. "Enhanced efficacy of DNA vaccination against Her2 / neu tumor antigen by genetic adjuvants"; IJC vol 111 pages 86-95, August 10, 2004

Pupa et al. "HER2: A biomarker at the crossroads of breast cancer immunotherapy and molecular medicine." JCP vol. 205 pages 10-18, May 10, 2005.

Quaglino E et al. "Concordant morphologic and gene expression data show that a vaccine halts HER2 / neu 25 preneoplastic lesions". JCI, vol. 113 No. 5 March 2004.

Bernhard H et al. "Vaccination against the HER2 / neu oncogenic protein". Endocrine-Related Cancer 9 (1) 33-44, 2002.

Berta, GN et al. "Anti-HER2 DNA vaccine protects Syrian hamsters against squamous cell carcinomas." Br J Cancer, vol 93 (11), November 28, 2005.

30 Disis et al. "Peptide-based, but not whole protein, vaccines elicit immunity to HER2 / neu, an oncogenic self-protein". The Journal of Immunology 156: 3151-3158, May 1, 1996.

Eck et al. ("Gene Based Therapy in The Pharmacological Basis of Therapeutics", Goodman and Gilman, Eds, 1996, p. 77-101).

Zhai et al. "Antigen-Specific Tumor Vaccines." The Journal of Immunology 156: 700-710, January 1996. Verma and 35 Somia. "Gene and therapy-promises, problems and prospects." Nature 389: 239-242, September 1997.

Miller and Vile. "Targeted vectors for gene therapy." FASeB J. 9: 190-199, 1995.

Deonarain, Mahendra. "Ligand-targeted receptor-mediated vectors for gene delivery". Exp. Opin. Ther. Patents 8 (1): 53-69, 1998.

Crystal "Transfer of Genes to Humans: Early Lessons and Obstacles to Success." Science 270: 404-410, October 20, 1995.

B. Bouchard et al., "Induction of Pigmentation in Mouse Fibroblasts by Expression of Human Tyrosinase cDNA", J. Exp. Med., 1989, vol. 169, p. 2029-2042.

B. Bouchard et al., "Production and Characterization of Antibodies against Human Tyrosinase", The Journal of Investigative Dermatology, 1994, vol. 102, No. 3, p. 291-295.

45 J. Rowell et al., "Lysosome-Associated Membrane Protein-1-Mediated Targeting of the HIV-1 Envelope Protein to an Endosomal / Lysosomal Compartment Enhances Its Presentation to MHC Class II-Restricted T Cells", The American Association of Immunologists , 1995, pag. 1818-1828.

S. Krishnan et al., "Paving the way towards DNA vaccines", Nature Medicine, 1995, vol. 1, n ° 6, pag. 521-522.

S. Barclay et al., "Rapid isolation of monoclonal antibodies specific for cell surface differentiation antigens", Proc. 50 Natl. Acad. Sci. USA, 1986 vol. 83, p. 4336-4340.

S. Vijayasaradhi et al., "Intracellular Sorting and Targeting of Melanosomal Membrane Proteins: Identification of Signals for Sorting of the Human Brown Locus Protein, GP75", The Journal of Cell Biology, 1995, vol. 130, n ° 4, pag. 807-820.

D. Pardoll et al., "Exposing the Immunology of Naked DNA Vaccines", Immunity, Cell Press, 1995, vol. 3, page 16555 169.

S. Vijayasaradhi et al., "The Melanoma Antigen gp75 is the Human Homologue of the Mouse b (Brown) Locus Gene Product", J. Exp. Med., 1990, vol. 171, p. 1375-1380.

G. Adema et al., "Molecular Characterization of the Melanocyte Lineage-specific Antigen gyp 100", The Journal of Biology Chemistry, The American Society for Biochemistry and Molecular Biology, 1994, vol. 269, No. 31, p. 2012660 20133.

A. Houghton et al., "Recognition of Autoantigens by Patients with Melanoma", Annals New York Academy of Sciences, 1993, p. 59-69.

C. Naftzger et al., "Immune response to a differentiation antigen induced by altered antigen: A study of tumor rejection and autoimmunity", Proc. Natl Acad. Sci. USA, 1996, vol. 93, p. 14809-14814.

F. Ausubel et al., "Expression of Proteins is Insect Cells using Baculoviral Vectors", Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, 1990, vol. 8, 16.8.1-16.11-7.

5 J. Ulmer et al., "Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein", Science, 1993, vol. 259, p. 1745-1749.

C. Tiffs et al., "The Folding and Cell Surface Expression of CD4 Requires Glycosylation", The Journal of Biological Chemistry, 1992, vol. 267, No. 5, p. 3268-3273.

S. Park, "JL1, A Novel Differentiation Antigen of Human Cortical Thymocyte", J. Exp. Med., The Rockefeller 10 University Press, 1993, vol. 178, p. 1447-1451.

C. Cabanas et al., "Characterization of a CD 11 c-Reactive Monoclonal Antibody (HCI / I) Obtained by Immunizing with Phorbol Ester Differentiated U937 Cells", Hybridoma, 1988, vol. 7, No. 2, p. 167-177.

N. Nanda et al. "Induction of Anti-Self-Immunity to Cure Cancer", Cell, 1995, vol. 82, p. 13-17, cited by others.

D. Peruzzi et al., Vaccine 28 (2010), p. 1201-1208 describes telomerase and HER-2 / neu as targets of genetically modified cancer vaccines in dogs.

SUMMARY OF THE INVENTION

[0006] It has now been found that the tolerance of the immune system by self-derived target differentiation antigens can be overcome and an immune response stimulated by the administration of a

xenogenic differentiation antigen (wild or mutant type) of the same type of a different species from the subject being treated (documents 6,328,969 and US 7,556,805 Sloan-Kettering).

[0007] For example, a rat differentiation antigen can be used to stimulate an immune response in the corresponding differentiation antigen in a canine subject. The administration of antigens

altered according to the invention results in an effective immunity against the original antigen expressed by the cancer in the treated subject. Therefore, according to a first aspect of the invention, a Her2 / neu xenogenic antigen is provided that is xenogenic of a Her2 / neu antigen expressed by dog mammary cells, for use in the treatment of canine carcinoma / breast tumor in a dog suffering from canine mammary carcinoma / tumor 30, in which the Her2 / neu xenogenic antigen is in an effective immunological amount and the Her2 / neu xenogenic antigen is administered after the surgical removal of tumors in subjects suffering from such cancers, and in that the Her2 / neu antigen associated with xenogenic mammary gland tumor is a vector comprising a DNA sequence encoding the Her2 / neu antigen associated with xenogenic therapeutic mammary gland tumor under the control of a promoter that promotes antigen expression Her2 / neu associated with 35 mammary gland tumor in the dog. There is also provided a xenogenic Her2 / neu antigen that is xenogenic of a Her2 / neu antigen expressed by dog mammary gland cells, for use in the treatment of canine mammary gland tumor in a dog suffering from canine mammary gland carcinoma / tumor , in which the Her2 / neu antigen associated with xenogenic mammary gland carcinoma / tumor is a vector comprising a DNA sequence encoding the Her2 / neu antigen associated with xenogenic therapeutic mammary mammary carcinoma / gland tumor under the control of a promoter that promotes the expression of the Her2 / neu antigen associated with xenogenic mammary gland tumor in dogs, and in which the vector has a sequence comprising 106-3885 of the sequence set forth in SEQ ID NO: 1 and the Her2 / antigen Neu xenogenico is administered after surgical removal of tumors in subjects suffering from such cancers. A vector is also provided which is capable of expressing in vivo in a canine the protein shown in SEQ ID NO: 2 for use in the treatment of canine carcinoma / mammary tumors in a dog suffering from canine carcinoma / mammary tumor, in the dog. that the canine mammary carcinoma / tumor is a canine mammary carcinoma / tumor associated with Her2 / neu, in which the vector is administered after the surgical removal of tumors in subjects suffering from such cancers. The use of a xenogenic Her2 / neu antigen that is xenogenic of a Her2 / neu antigen expressed by dog mammary cells is also provided for the manufacture of a medicament for the treatment of canine carcinoma / breast tumor in a dog suffering from carcinoma. / canine mammary tumor, in which the Her2 / neu xenogenic antigen is in an effective immunological amount and the Her2 / neu xenogenic antigen is administered after the surgical removal of tumors in subjects suffering from such cancers, and in which the Her2 antigen / neu associated with xenogenic mammary gland tumor is a vector comprising a DNA sequence encoding the Her2 / neu antigen associated with xenogenic therapeutic mammary gland tumor under the control of a promoter that promotes the expression of the associated Her2 / neu antigen a mammary gland tumor in dogs. As an additional aspect, the use of a xenogenic Her2 / neu antigen that is xengenic of a Her2 / neu antigen expressed by dog mammary gland cells is provided for the manufacture of a medicament for the treatment of canine mammary gland tumor in a dog suffering from canine mammary gland carcinoma / tumor, in which the Her2 / neu antigen associated with xenogenic mammary gland carcinoma / tumor is a vector that comprises a DNA sequence encoding the carcinoma / tumor associated Her2 / neu antigen of xenogenic therapeutic mammary gland under the control of a promoter that promotes the expression of the Her2 / neu antigen associated with xenogenic mammary gland tumor in dogs, and in which the vector has a sequence that

It comprises 106-3885 of the sequence set forth in SEQ ID NO: 1 and the Her2 / neu xenogenic antigen is administered after surgical removal of tumors in subjects suffering from such cancers. The scope of the present invention is defined by the claims. The subject matter outside the scope of the claims is for information only.

5

[0008] Therapeutic differentiation antigens based on differentiation antigens associated with carcinoma / tumor of mammary gland according to the invention are used to treat, for example, mammary gland carcinoma after surgical removal of tumors in subjects suffering from such cancers. . In one embodiment of the invention, a plasmid comprising a sequence encoding a receptor is administered to a subject.

Xenogenic tyrosine kinase, for example rat tyrosine kinase receptor, under the control of a suitable promoter. For example, dogs have been treated using plasmids comprising a DNA sequence encoding the rat tyrosine kinase receptor with a pronounced clinical benefit.

BRIEF DESCRIPTION OF THE DRAWINGS

fifteen

[0009]

FIG. 1A shows the overall survival time after immunization and surgical resection of MGT; FIG. 1B shows the disease-free survival time after immunization and surgical resection 20 of the MGT;

FIG. 1C shows metastasis-free survival time after immunization and surgical resection of MGT;

FIG. 2 shows a map of plasmid pcDNA3.1 (+/-);

FIG. 3 shows a map and the sequence of the human pING-tyrosinase plasmid, in which the human tyrosine coding sequence 25 has been removed. This is where rat Her2 / neu (nucleotides 17-3799 of SEQ ID NO: 1) was inserted to produce rHer2 / neu-pING of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides a method for treating mammary gland tumors in a

subject by stimulating an immune response to a differentiation antigen associated with mammary gland. The subject is preferably canine or feline, although the invention can be applied to other animal species, preferably mammalian or bird species as well.

[0011] As used in the specification and claims of this application, the term "response

immune ”encompasses both cellular and humoral immune responses. Preferably, the immune response is sufficient to provide immunoprotection against the growth of tumors that express the target differentiation antigen. The term "stimulates" refers to the initial stimulation of a new immune response or the potentiation of a pre-existing immune response.

40

[0012] According to the invention, a subject is treated by administering a xenogenic differentiation antigen of the same type as a target differentiation antigen expressed by mammary gland tumor cells of the subject in an amount effective to stimulate an immune response. . Therefore, the target differentiation antigen is the Her2 / neu antigen found in breast cells, and the therapeutic antigen

45 is a Her2 / neu xenogenic antigen.

[0013] In one embodiment, the use of the invention may include the following steps: (1) immunization of an animal in need of a xenogenic antigen, for example rat Her2 / neu exposed in SEQ ID NO: 2 and encoded by nucleotides 106-3885 of the sequence set forth in SEQ ID NO: 1, (2) needleless sensitization

50 of immune responses, (3) electrotransfer-based recall and (4) vaccination after tumor cytoreduction by primary surgical therapy.

[0014] In another embodiment, the use of the invention is carried out in subjects, including companion animals, without metastases (specifically, in relatively early stages of disease progression

55 breast carcinoma).

[0015] In some embodiments, the memory comprises administering plasmids encoding xenogenic antigens, for example those encoding rat Her2 protein (SEQ ID NO: 2).

[0016] In some embodiments, the xenogenic antigen is encoded by a nucleotide that has

Favorable nucleotide substitutions with respect to the sequence set forth in SEQ ID NO: 1. Favorable substitutions include any change that results in an improved immune response against Her2 / neu

expressed by tumor cells / breast carcinoma. Substitutions may include existing sequences such as murine Her2 (SEQ ID NO: 3), human Her2 (SEQ ID NO: 4) or any xenogenic Her2 sequence, or a fragment thereof, capable of triggering a therapeutically effective immune response in a target animal against a mammary carcinoma associated with Her2.

5

[0017] In some embodiments, the memory comprises administering a xenogenic differentiation antigen.

[0018] In other embodiments, the memory comprises administering a singen differentiation antigen.

10

[0019] The xenogenic differentiation antigen can be administered as a purified differentiation antigen derived from the source organism. The proteins can be purified for this purpose from cell lysates using column chromatography procedures. The proteins for this purpose can also be purified from recombinant sources, such as bacterial or yeast clones or mammalian or insect cell lines

15 expressing the desired product.

[0020] Administration of the xenogenic differentiation antigen can be accomplished by several routes. First, the xenogenic differentiation antigen can be administered as part of a vaccine composition that may include one or more adjuvants such as alumina, QS21, TITERMAX or its derivatives, adjuvant of

20 Incomplete or complete and related Freund and cytokines such as granulocyte-macrophage colony stimulating factor, ligand flt-3, interleukin 2, interleukin 4 and interleukin 12 to increase the intensity of the immune response. The vaccine composition may be in the form of a xenogenic differentiation antigen in a solution or suspension, or the therapeutic differentiation antigen may be introduced into a lipid carrier such as a liposome. Such compositions will generally be administered subcutaneously, intradermally or intramuscularly. Vaccine compositions containing expressed xenogenic differentiation antigen are administered in amounts that are effective to stimulate an immune response to the target differentiation antigen in the subject. The preferred amount to be administered will depend on the species of the subject and the specific antigen, but can be determined by routine preliminary tests in which increasing doses are procured and the extent of antibody formation or T-lymphocyte response is measured by ELISA or 30 tests. Similar. T lymphocyte responses can also be measured by cellular immune assays such as cytotoxicity, cytokine release assays and proliferation assays.

[0021] The xenogenic differentiation antigen can also be introduced according to the invention using a DNA immunization technique in which DNA encoding the antigen is introduced into the subject of such

35 so that the xenogenic differentiation antigen is expressed by the subject. CDNA encoding the differentiation antigen is combined with a promoter that is effective for the expression of the nucleic acid polymer in mammalian cells. This can be achieved by digesting the nucleic acid polymer with a restriction endonuclease and cloning it into a plasmid containing a promoter such as the SV40 promoter, the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) promoter. The resulting construct 40 is then used as a vaccine for genetic immunization. The nucleic acid polymer could also be cloned into plasmid and viral vectors that are known to transduce mammalian cells. These vectors include retroviral vectors, adenoviral vectors, Vaccinia virus vectors, poxvirus vectors and adenovirus associated vectors.

[0022] Nucleic acid constructs containing the promoter and the antigen coding region

they can be administered directly or they can be packaged in liposomes or coated on colloidal gold particles before administration. Techniques for packaging DNA vaccines in liposomes are known in the art, for example, in Murray, ed. "Gene Transfer and Expression Protocols" Humana Pres, Clifton, N.J. (1991). Similarly, techniques are taught to coat bare DNA on gold particles in Yang, "Gene transfer 50 into mammalian somatic cells in vivo", Crit. Rev. Biotech. 12: 335-356 (1992), and techniques are found for the expression of proteins using viral vectors in Adolph, K. ed. "Viral Genome Methods" CRC Press, Florida (1996).

[0023] For genetic immunization, vaccine compositions are preferably administered via

Intradermal, subcutaneous or intramuscular by injection or bombardment of gas-actuated particles, and are supplied in an amount effective to stimulate an immune response in the host organism. The compositions can also be administered ex vivo to blood or bone marrow derived cells (which include APC) using liposomal transfection, particle bombardment or viral infection (including coculture techniques). The cells treated in the subject are then reintroduced to immunize. Although it will be understood that the amount of material needed will depend on the immunogenicity of each individual construct and cannot be predicted a priori, the process of determining the appropriate dosage for any given construct is clear. Specifically, a series of dosages of increasing size are administered, starting from approximately

0.1 pig, and the resulting immune response is observed, for example by measuring the antibody titer using an ELISA assay, detecting the CTL response using a chromium release assay or detecting the TH response (helper T lymphocytes) using a cytokine release assay.

[0024] Once the tolerance is broken by administration of the xenogenic differentiation antigen,

Subsequent treatments with singenic differentiation can be used to maintain, and in some cases enhance, the immune response (see, Weber, et al., "Tumor immunity and autoimmunity induced by immunization with homologous DNA." J Clin Invest 102 (6): 1258 (1998)). Therefore, in one embodiment of the invention, the subject is first treated by administering a xenogenic differentiation antigen

10 (for example, during three cycles of treatment) and subsequently by administering a singen differentiation antigen (for example, for three additional cycles of treatment). As an alternative to treatment cycles using different therapeutic agents, a single therapeutic agent can be used that contains both xenogenic and singenic differentiation antigens. Therefore, for example, a mixture of rHer2-pING and hHer2-pING vectors, or a single vector encoding both rat and human Her2 / neu vectors can be used

15 under the control of a promoter so that they are expressed in a canine subject, for the treatment of mammary gland tumor in canines. There are commercially available vectors, for example in Stratagene and other companions, which can express two independent genes. Commonly, these vectors use an internal ribosome entry site, or IRES, between the two genes. This approach has the advantage of requiring the approval of only one therapeutic agent.

twenty

[0025] The invention will now be described further with reference to the following non-limiting examples.

Example 1- Construction of the Her2 / neu expression plasmid

[0026] The extracellular domain of rat HER2 / neu was amplified by PCR (nucleotides 17-3799 of SEQ ID

NO: 1) from plasmid pCMVneuNT (Amici et al., 1998) using the coding primers: 5'- CGAAGCTTACCATGGAGCTGGCGGCCTGG-3 '(SEQ ID NO: 6) and inverse: 5'-

CGGAATTCTTATGTCACCGGGCTGGC-3 '(SEQ ID NO: 7). The HindIII-EcoRI fragment was cloned into pcDNA3.1 (+) (Invitrogen, Carlsbad, CA; and FIG. 2). The original rat neu cDNA sequence has been described above.

30 (Bargmann et al., 1986), and is set forth herein in SEQ ID NO: 1, with the coding sequence of nucleotides 17 to 3799. The coding sequence of rat HER2 / neu is then subcloned in the pING vector (Bergman et al., Clin Cancer Res, 9: 1284-1290, 2003, skeleton depicted in FIG. 3; map depicted in FIG. 3A; and sequence set forth in SEQ ID NO: 5), procuring rat HER2 / neu-pING.

Example 2 - Immunization of positive canines of mammary gland tumor (MGT) with pING-rHer2

[0027] In this trial, 10 dogs with MGT were enrolled and immunized with 100 pg of pING- DNA

rHer2 per dose. The characterization for these dogs is shown in Table 1 and tumor staging is shown in Table 2.

40

 Age (years) Race Weight (kg)

 MGT 01

 9 Yorkshire terrier 1.75

 MGT 02

 13 Mixed 9.8

 MGT 03

 12 Yorkshire terrier 5

 MGT 04

 7 Lhasa Apso 11

 MGT 05

 10 Maltese 3.35

 MGT 06

 12 Cavalier King Charles Spaniel 9

 MGT 07

 8 Pomeranian 2.8

 MGT 08

 12 Maltese 3.9

 MGT 09

 13 Pomeranian 2.7

 MGT 10

 12 Yorkshire terrier 3

 Median

 12 3.6

Table 2. Tumor staging

 Tumor size (cm) Type of MGT Stage

 MGT 01

 2x2x4 0.2x0.2x0.2 0.2x0.3x0.2 0.1x0.1x0.1 0.5x0.5x0.5 0.2x0.2x0.2 0.5x0.5x0.5 T3N0M0 tubulopapillary carcinoma

 MGT 02

 12x10x8 5x3x1.5 1x1x1 1x1x0.5 0.5x0.1x0.1 Carcinoma rich in lipids T3N0M0

 MGT 03

 5.6x4.8x4.6 1.8x1.5x1.2 Tubulopapillary carcinoma with T3N0M0 fibroadenoma

 MGT 04

 4.2x5.6x2.5 T3N0M0 tubulopapillary carcinoma

 MGT 05

 1.2x1x0.5 1x1.4x0.5 1x1x0.4 0.5x0.5x0.5 Simple adenoma T1N0M0

 MGT 06

 10x4x3 Adenoma rich in lipids with T3N0M0 fibroadenoma

 MGT 07

 1x1x1 0.5x0.5x0.5 Complete type T1N0M0

 MGT 08

 1x1x1 0.5x0.5x0.5 Complex type T1N0M0

 MGT 09

 2.5x2x1 1.5x2x1 Complex type T1N0M0

 MGT 10

 1x1x1 0.5x0.5x0.5 0.1x0.1x0.1 T1N0M0 tubulopapillary carcinoma

[0028] As indicated, this group inclines five dogs in stage I and five in stage II, which all received

three doses of vaccine at two week intervals. The first and second doses were administered with the VITAJET ™ transdermal device and the third dose by intramuscular injection simultaneously with electroporation. Vaccination was initiated after the surgical withdrawal of MGT with simultaneous ovariohysterectomy (OHE). All dogs were negative for regional metastatic lymph node and lung. The disease-free and overall survival times were calculated using the day of surgery as day 0, with the results presented in Table 3.

Table 3. Disease-free and overall survival time.

 Dog

 WHO stage Disease-free survival Overall survival time (days) Clinical outcome

 Recurrence

 Metastasis

 MGT 05

 I 703 703 703 Live

 MGT 07

 I 669 669 669 Live

 MGT 08

 I 548 548 548 Live

 MGT 09

 I 536 536 536 Live

 MGT 10

 I 482 482 482 Dead

 Stage I Dogs

 548 548 548 -

 MGT 01

 III 779 779 779 Live

 MGT 02

 III 212 182 212 Dead

 MGT 03

 III 762 762 762 Live

 MGT 04

 III 575 381 720 Live with met.

 MGT 06

 III 686 686 686 Live

 Stage III dogs

 686 686 720

 Medium of all dogs

 622 609 678

15 [0029] A group of 19 dogs were identified as historical control cases. All control dogs

they experienced the surgical withdrawal of MGT with simultaneous OHE and were negative for regional metastases in lymph and pulmonary nodules. This group includes 7 dogs in stage I, 3 in stage II and 9 in stage III. Disease-free and overall survival times for these dogs were calculated using the day of surgery as day 0. The characterization of these dogs is shown in Table 4 and the tumor staging for each dog in the dog is exposed.

Table 5. Disease-free and overall survival times for the control group were calculated and presented in FIG 1A-1C.

Table 4. Characterization of the control dog

 Case number Age (years) Race Weight (kg)

 one

 9403460 7 Mixed 1.75

 2

 9404023 14 Poodle 2.5

 3

 9405132 14 Yorkshire 2.3

 4

 9409179 12 Finnish Spitz 6.8

 5

 9409043 14 Poodle 3.2

 6

 9500057 9 Lhasa Apso 6.5

 7

 9500890 14 Maltese 6

 8

 9500959 15 Cocker 14

 9

 923543 11 Siberian Husky 16

 10

 9405082 13 Poodle 3.9

 eleven

 9505202 9 Mixed 12

 12

 9600998 10 Maltes 4.6

 13

 9700451 13 Maltes 2.7

 14

 892285 12 Yorkshire 1.6

 fifteen

 9502927 14 Maltes 3.2

 16

 9405356 10 Cocker 12

 17

 9409104 11 Maltes 3.8

 18

 9503957 6 Miniature Schnauzer 4

 19

 9404023 14 Poodle 3

 Medium 12 3.9

5

Table 5. Tumor staging for control dogs

 Cnnico Tumor size Type of MGT Stage

 one

 9403460 6x6x7 Complementary carcinoma T3N0M0

 2

 9404023 3x3x3 Spinocellular carcinoma T2N0M0

 3

 9405132 7x4x7 2x2x2 0.3x0.2x0.2 0.5x0.5x0.5 Simple or complete carcinoma T3N0M0

 4

 9409179 13x12x12 6x7x7 1x1x1 Simple carcinoma with T3N0M0 squamous cell carcinoma

 5

 9409043 3.5x2.x1 3x1.5x1 T2N0M0 tubulopapillary carcinoma

 6

 9500057 3x2x2 2x1x1 T2N0M0 tubulopapillary carcinoma

 7

 9500890 8x3x1 Simple carcinoma T3N0M0

 8

 9500959 8x3x2 2x1x0.5 T3N0M0 adenocarcinoma

 9

 923543 5x5x4 0.2x0.2x0.2 Simple Carcinoma T3N0M0

 10

 9405082 5x4x3.5 3x3.5x3 Simple carcinoma T3N0M0

 eleven

 9505202 0.3x0.3x0.3 1x1x0.5 0.4x0.4x0.4 T1N0M0 tubulopapillary carcinoma

 12

 9600998 0.5x0.5x0.4 1x0.5x0.5 Carcinoma T1N0M0

 13

 9700451 1x1x1 1x1x1 T1N0M0 tubulopapillary carcinoma

 14

 892285 0.5x0.8x0.3 1x0.8x0.5 Carcinoma in benign mixed tumor T1N0M0

 fifteen

 9502927 5x4x4 0.5x0.5x0.5 Carcinoma in benign mixed tumor T3N0M0

 16

 9405356 10x3x1.5 T3N0M0 tubulopapillary carcinoma

 17

 9409104 1x1x1 0.5x0.5x0.5 2x2x2 Adenocarcinoma T1N0M0

 18

 9503957 2x2x2 0.3x0.3x0.3 Adenocarcinoma, complete type T1N0M0

 19

 9404023 2x2x1 Adenocarcinoma T1N0M0

[0030] Philibert et al. (2003) reviewed survival statistics for 97 dogs with MGT and reviewed

that the median survival times for 41 dogs with MGT less than 3 cm in diameter were 22 months

10 (~ 666 days) versus 14 months (~ 424 days) for 56 dogs with MGT larger than 3 cm in diameter. In the absence of lymph node involvement or metastasis, a tumor size smaller than 3 cm correlates with stage I disease and greater than 3 cm correlates with a stage II or greater pathological state. They found no difference in survival time for dogs in stages II, III or IV.

[0031] The median overall survival time for all dogs treated with pING-rHer2 vaccine

It is 678 days. This was significantly higher compared to the historical data of the 19 dogs provided by NTU, which indicate a median overall survival time of 300 days, and with data published by Philibert et al. (2003), which indicate 424 days of overall survival time for dogs with

Stage II MGT or greater.

[0032] The pING-rHer2 DNA vaccine will target dogs and cats with tumors that are shown to

overexpress Her2 antigen based on tumor tissue analysis, using existing 5 Her2 tissue expression assays. The vaccine will be administered using the Vetjet ™ transdermal device to deliver 100 pg of DNA to the medial thigh of dogs or to the lateral thigh of cats, at two-week intervals for four doses. Surviving dogs and cats will receive a souvenir dose every 6 months.

Claims (9)

1. A xenogenic Her2 / neu antigen that is xenogenic from a Her2 / neu antigen expressed by mammary cells of a dog, for use in the treatment of canine mammary carcinoma / tumor in a dog suffering from 5 Canine mammary carcinoma / tumor, where the xenogenic Her2 / neu antigen is in an immunologically effective amount and the xenogenic Her2 / neu antigen is administered after the surgical removal of tumors in subjects suffering from such cancers, and where the Her2 / neu antigen Xenogenic mammary gland tumor associated is a vector that comprises a DNA sequence encoding the Her2 / neu antigen associated with xenogenic therapeutic mammary gland tumor under the control of a promoter that promotes tumor associated Her2 / neu 10 expression of mammary gland in dog. 2. The xenogenic Her2 / neu antigen of claim 1 for use according to claim 1, wherein the differentiation antigen associated with xenogenic mammary gland tumor is rat Her2 / neu. 15 3. A xenogenic Her2 / neu antigen that is xenogenic from a Her2 / neu antigen expressed by Mammary gland cells of a dog, for use in the treatment of canine mammary gland tumor in a dog suffering from canine mammary gland carcinoma / tumor, where the Her2 / neu antigen associated with xenogenic mammary gland carcinoma / tumor is a vector comprising a DNA sequence encoding a Her2 / neu antigen associated with xenogenic therapeutic mammary gland carcinoma / tumor under the control of a promoter that promotes the expression of the Her2 / neu antigen associated with xenogenic mammary gland tumor in dogs, and where the vector has a sequence comprising 106-3885 of the sequence set forth in SEQ ID NO: 1 and the antigen Her2 / neu xenogenic is administered after surgical removal of tumors in subjects suffering from such cancers. 25 4. The xenogenic Her2 / neu antigen according to claim 1 or 2 for use in accordance with the claim 1 or 2, where: 1) Her2 / neu associated carcinoma is surgically cytoreducted; 2) a sensitizing immunization comprising a first plasmid encoding a xenogenic Her2 / neu antigen is administered; Y 3) an immunization is administered by electrotransfer / electroporation; where the memory is the first plasmid or is a second plasmid capable of expressing in vivo in a canine a different Her2 / neu xenogenic antigen, including those encoded by SEQ ID NO: 3 or 4, or is a recombinant vector capable of expressing in I live any Her2 / neu protein that is capable of triggering a therapeutically effective immune response against heterologous Her2 / neu expressed by Her2 / neu associated carcinoma. 35 5. The xenogenic Her2 / neu antigen according to claim 4 for use according to claim 4, wherein: 1) sensitizing immunization is administered without a needle; 2) the first plasmid is capable of expressing in vivo in a canine a sequence set forth in SEQ ID NO: 2; 40 3) booster immunization comprises administration of the plasmid of stage 2. 6. The xenogenic Her2 / neu antigen according to claim 4 or 5 for use according to claim 4 or 5, wherein the immunization of souvenir to the surviving canines is provided once every 3 to 6 months. Four. Five 7. The xenogenic Her2 / neu antigen according to any one of claims 1 or 2 for use according to claim 1 or 2, wherein the xenogenic differentiation antigen is administered by immunization with DNA of the subject with DNA encoding the xenogenic differentiation antigen in a non-viral plasmid vector comprising DNA encoding the xenogenic differentiation antigen under the control of a 50 promoter that promotes the expression of the xenogenic differentiation antigen. 8. The xenogenic Her2 / neu antigen according to any one of claims 1 or 2 for use according to any one of embodiments 1, 2 or 7 performed simultaneously to the resection of a mammary gland tumor (MGT). 55 9. A vector that is able to express in vivo in a canine the protein exposed in SEQ ID NO: 2, for use in the treatment of canine carcinoma / mammary tumors in a dog suffering from canine mammary carcinoma / tumor, where the carcinoma / Canine mammary tumor is a carcinoma / mammary tumor associated with Her2 / neu, where the vector is administered after surgical removal of tumors in subjects suffering from such cancers. 60 10. Use of a xenogenic Her2 / neu antigen that is xenogenic of a Her2 / neu antigen expressed by a dog's mammalian cells, for the manufacture of a medicament for the treatment of carcinoma / tumor canine mammary in a dog suffering from canine carcinoma / tumor, where the xenogenic Her2 / neu antigen is in an immunologically effective amount and the xenogenic Her2 / neu antigen is administered after the surgical removal of tumors in subjects suffering from such cancers, and where the Her2 / neu antigen associated with xenogenic mammary gland tumor is a vector that comprises a DNA sequence encoding the 5 Her2 / neu antigen associated with xenogenic therapeutic mammary gland tumor under the control of a promoter that promotes antigen expression Her2 / neu associated with mammary gland tumor in dogs. 11. Use of a xenogenic Her2 / neu antigen that is xenogenic of a Her2 / neu antigen expressed by Mammary gland cells of a dog, for the manufacture of a medicament for the treatment of tumor of 10 canine mammary gland in a dog suffering from canine mammary gland carcinoma / tumor, where the Her2 / neu antigen associated with carcinoma / gland tumor Xenogenic mammary is a vector that comprises a DNA sequence encoding the Her2 / neu antigen associated with xenogenic therapeutic mammary gland carcinoma / tumor under the control of a promoter that promotes the expression of the Her2 / neu antigen associated with xenogenic mammary gland tumor in dogs, and where the vector has the sequence comprising 106-3885 of the sequence 15 set forth in SEQ ID NO: 1 and the xenogenic Her2 / neu antigen is administered after surgical removal of tumors in subjects suffering from such cancers.